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Type I allergic diseases such as seasonal allergic rhinitis (hayfever), conjunctivitis, allergic asthma and allergic dermatitis represent a major health problem in industrialized countries (Wuthrich, Int. Arch. Allergy Immunol. 90: 3-10, 1989). It is currently estimated that 15-20% of the population in developed countries are afflicted with some form of allergy (Miyamoto, Advances in Allergology and Clinical Immunology. Godard P, Bousquet J, Michel F B (eds) pp. 343-347. The Parthenon Publishing Group, Cornforth, UK, 1992). Therefore, the diagnosis and therapy of these diseases have become focal points of interest for scientific investigation.
The primary immunological and biochemical bases of type I allergic reactions are the interaction of allergenic substances (allergens) with IgE antibodies bound to high affinity Fc receptors on the surface of mast cells and basophils. This interaction results in cross linking of allergen-specific IgE antibodies which in turn stimulates an immediate release and cascade production of inflammatory mediators responsible for allergic symptoms. Allergens are present in airborne particles such as house-dust, pollen of grasses, weeds and trees, mould spores and animal dander.
At present, one form of therapeutic intervention of allergic diseases (such as rhinitis, conjunctivitis and allergic asthma) involves injection of the allergen assumed to be responsible for the allergic response. This is referred to as hypo-sensitization treatment. Extracts currently in use in this procedure are prepared from natural sources and contain, in addition to the allergens, components such as proteins to which patients are not allergic.
The development of recombinant techniques has provided the means to produce high levels of purified allergens for diagnostic and therapeutic purposes. However, the high level of purity of recombinant allergen preparations results in a high anaphylactogenic index even at very low doses. Accordingly, extreme care is required when they are administered to patients. There is a need, therefore, to develop recombinant allergens with a reduced risk of anaphylactic shock.
The major outdoor cause of seasonal hay-fever and allergic asthma is airborne grass pollen (Smart et al., Int. Arch. Allergy Immunol. 7: 243-248, 1983). Pollen calendars show that grass pollen is most abundant in spring and early summer when grasses flower and this is when allergic asthma peaks in incidence. The most important sources of grass pollen are common agricultural pasture grasses which have been widely introduced throughout the world, but vary in temperature and tropical climate zones. In cool temperature regions, grasses such as rye-grass, Kentucky bluegrass and timothy (all belonging to the subfamily Pooideae) are of clinical significance, whereas in warm temperature and subtropical environments pollen of Bermuda grass (subfamily Chloridoideae) becomes the most important source of allergens. The most comprehensive studies have been made on proteins from rye-grass pollen and to a lesser extent Kentucky bluegrass and timothy.
Individuals sensitive to allergens from one grass are often sensitive to those of a number of other grass genera. This is particularly true for pollen of grasses within the subfamily Pooideae (Smith et al., “Analysis of rye-grass pollen allergens using two dimensional electrophoresis and immunoblotting.” In Kraft D (ed), Molecular Biology and Immunology of Allergens, CRC Press, Boca Raton, Fla., 1994), where immunological cross-reactivity has been demonstrated in inhibition experiments using an IgE-binding assay, the radioallergosorbent test (RAST). In these experiments, pollen extracts from one grass were able to inhibit binding of IgE to extracts from other grasses.
Allergenic components of grass pollen can be classified into different groups according to their physiochemical and immunological properties. The major allergens that elicit an allergic reaction to pollen from the Pooid grasses are group 1 and 5 allergens, as judged by both criteria of the number of allergic patients responding and relative amounts of IgE binding to the allergens (Singh et al, 1991, supra). In case of perennial rye-grass, Lolium perenne, pollen extracts contain more than 17 proteins which have the capacity to bind IgE from sera of grass pollen allergic patients (Smith et al., 1993, supra). However, it has been shown that allergens of group 1 and 5 together can inhibit most of the IgE binding to crude pollen extracts (Bond et al., J. Allergy Clin. Immunol. 91: 339, 1993).
Lol p 5, a protein of 28-33 kDa, is the second most prevalent rye-grass allergen which causes allergy in 85-90% of grass pollen allergic individuals. Molecular cloning of cDNAs encoding this group 5 allergen (Singh et al., Proc. Natl. Acad. Sci. USA 88: 1384-1388, 1991; Ong et al., Gene 134: 235-240, 1993) has shown that Lol p 5 exists as a family of homologous but distinguishable isoforms which retain their IgE reactivity even after separation on denaturing SDS-PAGE gels and immunoblotting (Singh et al., 1991, supra).
A need, accordingly, exists to develop modified forms of recombinant allergens useful in immunotherapy and immunoprophylaxis of allergic conditions.